The present application represents U.S. national stage of international application PCT/SE99/00598 with an international filing date of Apr. 15, 1999. The international application was published in English under Article 21(2) of the PCT on November 4, 1999. It claims priority to Swedish application 9801455-8, filed on Apr. 24, 1988.
The present invention is in the general field of biological receptors and the various uses that can be made of such receptors. More specifically, the invention relates to nucleic acids encoding a novel neurotensin-like receptor (NLR) and to the receptor itself.
G protein-coupled receptors (GPCRs) constitute a family of proteins sharing a common structural organization characterized by an extracellular N-terminal end, seven hydrophobic alpha helices putatively constituting transmembrane domains and an intracellular C-terminal domain. GPCRs bind a wide variety of ligands that trigger intracellular signals through the activation of transducing G proteins (Caron, et al., Rec. Prog. Horm. Res. 48:277-290 (1993); Freedman et al., Rec. Prog. Horm. Res. 51:319-353 (1996)).
More than 300 GPCRs have been cloned thus far and it is generally assumed that there exist well over 1000 such receptors. Mechanistically, approximately 50-60% of all clinically relevant drugs act by modulating the functions of various GPCRs (Cudermann, et al., J. Mol. Med. 73:51-63 (1995)). Of particular interest are receptors located in the central nervous system. G protein-coupled receptors located in this region are known to be involved in the transmission, modulation and sensation of pain. Thus, new G protein-coupled receptors found in the brain and spinal column may be used in assays for the identification of new agents for producing anesthesia and analgesia.
The present invention is based upon the discovery of a novel G protein-coupled receptor which is expressed in the central nervous system and has a structure distinct from all previously reported receptors. Since it appears to share a substantial homology with the human neurotensin receptor, it is referred to herein as the xe2x80x9cneurotensin-like receptor.xe2x80x9d
In its first aspect, the invention is directed to a protein, except as existing in nature, comprising an amino acid sequence consisting functionally of SEQ ID NO:1. The term xe2x80x9cconsisting functionally ofxe2x80x9d refers to proteins in which the sequence of SEQ ID NO:1 has undergone additions, deletions or substitutions which do not substantially alter the functional characteristics of the receptor. The term is intended to encompass proteins having exactly the same amino acid sequence as that of SEQ ID NO:1, as well as proteins with sequence differences that are not substantial as evidenced by their retaining the basic, qualitative ligand binding and physiological properties of the neurotensin-like receptor. The term xe2x80x9cexcept as existing in naturexe2x80x9d refers to a compound that is either expressed by recombinant means or that is in a purified (preferably substantially purified) state.
The invention also encompasses a protein, except as existing in nature, having an amino acid sequence consisting essentially of the sequence of SEQ ID NO:1; antibodies that bind preferentially to such a protein (i.e., antibodies having at least a 100-fold greater affinity for NLR than any other protein); and antibodies made by a process involving the injection of a pharmaceutically acceptable preparation of NLR into an animal capable of antibody production.
In a preferred embodiment, monoclonal antibody to NLR is produced by administering, preferably by injection, NLR to a mouse and then fusing the mouse""s spleen cells with myeloma cells.
The invention is also directed to a polynucleotide, except as existing in nature, encoding a protein comprising an amino acid sequence consisting functionally of SEQ ID NO:1. This aspect of the invention encompasses polynucleotides encoding proteins consisting essentially of the amino acid sequence of SEQ ID-NO:1, expression vectors comprising such polynucleotides, and host cells transformed with such vectors. Also included is the recombinant neurotensin-like receptor produced by host cells made in this manner.
Preferably, the polynucleotide encoding the neurotensin-like receptor has the nucleotide sequence shown in SEQ ID NO:2, and the vectors and host cells used for expression of the receptor also use this particular polynucleotide.
In another aspect, the present invention is directed to a method for assaying a test compound for its ability to bind to a human neurotensin-like receptor. The method is performed by incubating a source of NLR with a ligand known to bind to the receptor and with the test compound. The source of receptor should, preferably, express a large amount of NLR relative to other G protein-coupled receptors. Upon completion of incubation, the ability of the test compound to bind to NLR is determined by the extent to which ligand binding has been displaced. Preferably, the receptor present should have the sequence shown in SEQ ID NO:1. Although not essential, the binding assay can be accompanied by an assay to determine whether a second messenger pathway, e.g., the adenyl cyclase pathway, has become activated. This should help to determine whether a particular compound binding to NLR is acting as an agonist or antagonist.
An alternative method for determining if a test compound is an NLR agonist, a method that does not require any ligand, is to use a cell signaling assay, e.g., an assay measuring either intracellular adenyl cyclase activity or intracellular calcium concentration. The test compound should generally be incubated with cells expressing high amounts of NLR relative to other G protein-coupled receptors, typically a cell transfected with an expression vector encoding the NLR of SEQ ID NO:1. Test compounds that are agonists are identified by their causing a statistically significant change in the results obtained from the cell signaling assay when compared to control cells not exposed to test compound. The control cells may be either cells that have not been transfected or cells that have been mock transfected with a vector that does not produce active receptor. NLR-expressing cells exposed to test compounds that are agonists would typically be expected to show a significant increase in adenyl cyclase activity or in intracellular calcium concentration relative to control cells.
The invention also encompasses a method for determining if a test compound is an antagonist of NLR which relies upon the known constitutive activation of G protein-coupled receptors that occurs when such receptors are expressed in large amounts. This method requires that DNA encoding the receptor be incorporated into an expression vector so that it is operably linked to a promoter and that the vector then be used to transfect an appropriate host. In order to produce sufficient receptor to result in constitutive receptor activation (i.e., activation in the absence of natural ligand), expression systems capable of copious protein production are preferred, e.g., the NLR DNA may be operably linked to a CMV promoter and expressed in COS or HEK293 cells. After transfection, cells with activated receptors are selected based upon their showing increased activity in a cell signaling assay relative to comparable cells that have either not been transfected or that have been transfected with a vector that is incapable of expressing functional NLR. Typically, cells will be selected either because they show a statistically significant change in intracellular adenyl cyclase activity or in intracellular calcium concentration. The selected cells are contacted with the test compound and the cell signaling assay is repeated to determine if this results in a decrease in activity relative to selected cells that have not been contacted with the test compound. For example, a statistically significant decrease in either adenyl cyclase activity or calcium concentration relative to control cells would indicate that the test compound is an antagonist of NLR. Preferably the NLR used in assays has the sequence of SEQ ID NO:1.
Assays for compounds interacting with NLR may be performed by incubating a source containing the receptor (e.g., a stably transformed cell) with a ligand specific for NLR both in the presence and absence of test compound and measuring the modulation of intracellular calcium concentration. A significant increase or decrease in ligand-stimulated calcium signaling in response to test compound is indicative of an interaction occurring at the neurotensin-like receptor. The preferred receptor is that having the amino acid sequence of SEQ ID NO:1.
In another aspect, the present invention is directed to a method for assaying a test compound for its ability to alter the expression of NLR. This method is performed by growing cells expressing NLR in the presence of the test compound. Cells are then collected and the expression of NLR is compared with expression in control cells grown under essentially identical conditions but in the absence of test compound. The preferred receptor is one having the amino acid sequence of SEQ ID NO:1. A preferred test compound is an oligonucleotide at least 15 nucleotides in length comprising a sequence complementary to the sequence of the NLR mRNA used in the assay.